Background and Significance Expression of HER2 receptors in breast cancers is correlated with poor prognosis and their expression may be different in distant metastases as compared to the primary tumor. The other target would be EGFR. Although a significant progress has been made in the understanding of the biology of ER-positive and HER2-positive breast cancers, which has led to the enormous growth in targeted therapeutic for this group of patients, few such developments have been made for ER/PR/HER2 triple negative breast cancer, which represent 10-17% of breast cancers that preferentially affect young and African-American women. The tumors frequently (56-86%) express epidermal growth factor receptor (EGFR) and, like HER2 overexpressing tumors, are associated with aggressive behavior and the poorest prognosis of all breast cancer subtypes with early relapses within the first five years. Although not synonymous, the majorities of TNBC carry the basal-like molecular profile on gene expression arrays and mostly are characterized by high p53 IHC expression or p53 gene mutations, which may have implications for chemotherapy sensitivity. Current data suggest that the majority of TNBC patients will be refractory or show only a partial response to conventional chemotherapy regimens and will die of metastatic disease within the first few years after diagnosis. It is noteworthy that, unlike for other subtypes, chemotherapy is currently the only modality of systemic therapy for patients bearing triple negative breast tumors and there is no clinically validated targeted therapy for their treatment. Therefore, the development of novel targeted therapies for this subset of high-risk patients is of paramount importance. Therefore, we propose a two-pronged research project that will utilize functional imaging for visualization and quantification of EGFR and therapeutic recombinant protein combining EGFR-Affibody and modified p53 tumor suppressor for targeted therapy to improve outcome of TNBC treatment. This project will provide means to assess global expression of HER2 or EGFR in breast cancers (including metastases) and to deliver therapeutic agents specifically to HER2 and EGFR-positve cells. As the targeting agent we propose to use Affibody molecules obtained from our CRADA partner in Sweden (http://www.affibody.com). These very stable and highly soluble alpha-helical proteins are relatively small (8.3 kDa) and can be readily expressed in bacterial systems or produced by peptide synthesis. The His6-Zher2:324 binds to HER2 receptors with high affinity (22 pM) and is available with cystein at the carboxy-terminal to facilitate conjugation. For imaging purposes, these molecules are labeled with radionuclides or near-infrared optical beacons. For therapy, to eradicate widespread disease in the metastatic setting (in which even the disease with known locations may be too widespread to use local treatment modalities) and for treatment in the adjuvant setting to treat non-detectable micrometastases, we are developing Affitoxins affibody-based recombinant proteins combining HER2-targeting capacity of Affibody with proteins having therapeutic potential such as, for example, bacterial toxins or pro-apoptotic cell signaling molecules. Initially, focus on development of proteins with optimal of subcellular localization capacity using Affiprobes- molecules containing fluorescent proteins. Then we will test appropriate effectors, for instance, PE38 and p53 for molecules localizing in cytosol and nucleus, respectively. Our strategy, involving assessment of target presence and distribution in an individual patient followed by optimized, target-specific drug delivery, may significantly improve efficacy of breast cancer treatment while reducing side effects. Research Design Labeling with imaging agents The Affibody molecules are conjugated using maleimide chemistry with either near-infrared fluorescent molecules or positron emitters for, respectively, optical and PET imaging. Affitoxins A recombinant DNA construct combining HER2-specific Affibody molecules with Pseudomonas toxin hgave been developed and cloned by PCR amplification of the PE38 part of HA22 immunotoxin following by ligation of the PCR product into vector containing the HER2-affibody under control of IPTG-inducible T7 promoter. Similar procedures will be used to produce recombinant proteins containing different effectors and sub-cellular localization domains. In vitro and in vivo characterization Using binding, proliferation and clonogenic survival assays, as well as molecular biology methods we will thoroughly characterize in vitro the binding properties of the conjugates and their effects on the target cells. Biodistribution of the novel tumor-targeted molecules will be studied using nude mice bearing xenografts of HER2-positive tumors. To test the in vivo imaging capacity of radioconjugates, we are monitoring, by optical and PET imaging, the downregulation of HER2 in tumor xenografts following treatment with Herceptin or DMAG. The estimation of expression level obtained from imaging data is verified by ex-vivo analysis of tumor tissue by immunohistochemistry and Western blots. The subcellular distribution of Affitoxin is tested using confocal microscopy and their therapeutic efficacy is assessed in vitro and in vivo. Accomplishments: 1. We have successfully labeled HER2-Affibody with a positron emitter, 68-Ga and validated the resulting tracer in vitro and in vivo. The results were published in European Journal of Nuclear Medicine and Molecular Imaging. 2. We have proved that AlexaFluor-labeled Affibody molecules can be used for in vivo quantification of changes in HER2 expression following therapeutic intervention. The results of this work have been published in Molecular Imaging. 3. We have genetically fused HER2-specific Affibody molecule with a truncated and optimized version of Pseudomonas Exotoxin A (PE38KDEL). The resulting recombinant protein called HER2-Affitoxin combines high HER2 specificity and affinity of Affibody molecules with the tumoricidal potential of PE38KDEL. The results of this work were published in Clinical Cancer Research and were featured in a recent pres release from American Association for Cancer Researchhttp://www.aacr.org/home/public--media/aacr-in-the-news.aspx?d=2421. 4. We have created photo-stable and relatively simple-to-produce imaging probes for in vitro staining of EGFR and HER2. These new reagents, called Affiprobes, consist of a targeting moiety, a HER2- or EGFR-specific Affibody molecule, and a fluorescent moiety, mCherry (red) or EGFP (green). Our flow cytometry and confocal microscopy experiments demonstrated high specificity and signal/background ratio of Affiprobes. Affiprobes are able to stain both live cells and frozen tumor xerograph sections. This type of optical probe can easily be extended for targeting other cell-surface antigens/receptors. The results of this work were published in European Journal of Chemical Biology: ChemBioChem. 5. Several Affibody-based recombinant proteins containing fluorescent proteins and different sub-cellular localization domains have been developed and their trafficking studies by confocal microscopy. 6. We have successfully applied 18-F-Labeled Affibody in multimodality imaging setting combining PET, CT and MRI, to detect beast micrometastases in the lungs. The resulting manuscript has been submitted to Clinical Cancer Research. 7. We have successfully applied 18-F-Labeled Affibody to study in vivo changes of HER2-expression during treatment with trastuzumab and showed that those changes might be used to predict early tumor response to trastuzumab. The resulting manuscript has been submitted to Clinical Cancer Research.